Signaling between osteoclasts (the cells that resorb bone and teeth) and osteoblasts (the cells that form bone) regulates normal and pathological bone and root resorption. Exosomes are small (30-120 nm in diameter) extracellular vesicles involved in cell-cell signaling that are being explored as potential disease markers. They are released from cells and interact with target cells, either at the cell surface or after being internalized, to affect the target cell's activity. Proteomic analysis of changes in the protein content of gingival crevicular fluid (GCF) associated with root resorption in humans by mass spectroscopy identified many proteins that have been found in exosomes. Osteoclasts, the cells directly responsible for root resorption, were then shown to release exosomes when cultured in vitro. The composition of the exosomes from osteoclasts changed when osteoclasts were actively resorbing bone, and osteoclast-derived exosomes stimulated osteoclast formation when added to calcitriol-stimulated mouse marrow cultures, which contain both osteoclasts and osteoblasts. These data inspired the central hypothesis of this proposal: Exosomes released by osteoclasts are involved in the regulation of bone remodeling and serve as markers for normal and pathological bone and tooth resorption. The goals of this low risk/high reward proposal are to test this hypothesis in cell culture by developing techniques to isolate subsets of exosomes released by osteoclasts, to study the composition of the isolated exosomes, and to test their biological activities. This can help build a framework for studying exosomes' role in regulating bone remodeling and for finding ways to use exosomes as biological markers for root and bone resorption. The first aim of this project is to isolate and characterize exosomes released from osteoclasts in vitro. Affinity chromatography using antibodies (previously used in exosome isolation) and lectins (novel), together with standard exosome isolation techniques will isolate subsets of osteoclast-derived exosomes with different compositions. The isolated exosomes will be characterized by electron microscopy and one and two- dimensional mass spectroscopy. Preliminary data suggest that exosomes from resorbing osteoclasts may have unique composition and activities. The second aim is to test the regulatory activity of the osteoclast- derived exosomes on primary calvarial osteoblasts. Exosomes from osteoclasts will be added to separate cultures of and mouse calvarial osteoblasts. The effects of exosomes on osteoblasts will be examined in well- characterized assays of cell differentiation, survival, activation, and activity. The team of researchers has extensive experience in all of the proposed methods. Successful completion of this project will provide a basis for novel studies of the role of exosomes in regulating bone remodeling. Because no reports in literature have studied exosomes in this context, our proposed work should provide new fundamental data that will lead to 1) innovative therapeutic modalities to treat dental diseases and 2) future translational effort using exosomes in GCF as biomarkers for the early detection of root resorption and pathological alveolar bone resorption.